Biochemical analysis of the Xenopus laevis TCR/CD3 complex supports the "stepwise evolution" model

The TCR/CD3 complex of a cold-blooded vertebrate, the amphibian Xenopus lae vis, was biochemically characterized with a cross-reactive polyclonal antis erum recognizing a conserved epitope in the cytoplasmic domain of CD3 epsil on. The specificity and utility of this reagent was validated by Western bl ot analysis and immunoprecipitation of the well-characterized chicken TCR/C D3 complex. Cross-reactivity with the X. laevis CD3 epsilon protein was dem onstrated by specific staining of sorted CD8(+) cells. Immunohistology on b oth tadpoles and adult tissues suggests this antiserum will be instrumental in the localization of Xenopus T cells and most likely NK cells. Double st aining of tissue sections with an anti-CD8 monoclonal antibody confirmed th at this staining is specific. The antiserum was also used for the biochemic al analyses of X. laevis TCR/CD3 complex. The 75-kDa alpha beta TCR heterod imer could be separated into a 40-kDa acidic TCR alpha chain and a 35-kDa b asic TCR beta chain. Two CD3 proteins, both comigrating at approximately 19 kDa, were associated with the TCR heterodimer. Removal of N-linked carbohy drates yielded CD3 proteins of 19 kDa and 16.5 kDa, most likely representin g the CD3 epsilon and CD3 gamma/delta homologues, respectively. An addition al band of 110 kDa represents a multimeric complex of the TCR heterodimer c ovalently linked to a CD3 dimer. These properties of the Xenopus TCR/CD3 co mplex substantiate a stepwise evolutionary model for the CD3 protein family .